A large number of non-natural analogues of DNA nucleosides have been synthesized in recent years. Changing the structure of the base moiety attached to deoxyribose has been a useful strategy for probing structure and function in DNA. For example, a number of base analogues have been used to test the importance of specific hydrogen bonding interactions which may be important for function of the natural nucleic acid bases..sup.1-3 Using this strategy, the importance of hydrogen bonding in stabilizing DNA and RNA structure, in protein interactions and in the fidelity of enzymatic DNA and RNA synthesis has been examined.
Modified DNA bases have also been synthesized with the purpose of serving as reporter groups in physical and biochemical studies of structure and function. Examples of reporter groups which have been attached to DNA bases include biotin.sup.4 and digoxigenin groups.sup.5, spin-label groups.sup.6, and DNA-cleaving moieties.sup.7. Among the most prominent classes of reporters used in DNA are fluorescent-tagged DNA bases which can serve as probes in biophysical and biochemical studies.sup.8. In contrast to placement of such reporter groups at the end of a DNA strand using nonnucleotide linkers, the attachment of a reporter to a DNA base allows for placement and probing within a stretch of DNA. Such a strategy has found considerable practical use in fluorescence-based automated DNA sequencing..sup.9 An alternative approach to the conjugation of a fluorophore to a natural DNA base is the more direct modification of a DNA base itself to render it fluorescent. A number of modified DNA bases with useful fluorescence properties have been reported recently; among the most widely used nucleosides of this type are 2-aminopurine.sup.10 and ethenoadenosine.sup.11.
DNA may be fluorescently tagged either enzymatically or synthetically. Enzymatic incorporation is carried out by use of nucleoside triphosphates carrying a given fluorophore. Incorporation into DNA by chemical methods is especially common. Chemical methods of incorporating fluorescent reagents into DNA is done by either of two methods: direct incorporation of a label which has been converted to a phosphoramidite derivative or incorporation of an amine into the oligonucleotide, followed by later derivatization with a fluorophore isothiocynate or NHS ester derivative.
The postsynthesis derivatizaton of DNA is typically inefficient, and requires steps beyond those of standard DNA synthesis as well as laborious purification steps. Direct incorporation of a label into DNA is attractive because standard DNA synthesis and purification steps are used. However, currently available reagents are quite expensive due to their cost of synthesis.
Another drawback to currently used fluorescent-tagged nucleic acids is the quenching phenomenon which occurs when multiple fluorescent tags are placed near each other. Thus, a nucleic acid with multiple fluorescent tags is often no brighter or even less bright than a nucleic acid with a single fluorescent tag.
In addition, it is commonly observed that a fluorescent tag is quenched by the act of attaching it to DNA. For example, it has been reported (Netzel, 1989 J. Am. Chem. Soc. 111:6966) that pyrene tags attached to a linker at the end of a DNA strand were quenched greatly (50-fold) in the DNA. Moreover, on binding a complementary sequence, the emission was quenched another ten-fold. This effect necessarily leads to lower sensitivity of detection.
Another limitation of commercially available fluorescent labels include their rapid photobleaching characteristics. As in fluorescence microscopy or blot hybridization, the practical brightness of a label depends on the time of integration of the emission signal. Fluorescein, for example, photobleaches quite rapidly in a DNA oligonucleotide, because of its complex structure (allowing greater reactivity) and because it is exposed to solution where more reactions occur. In addition, a label such as fluorescein is typically attached to DNA by flexible tethers. Measuring protein-DNA binding by time-resolved fluorescence an isotropy is often problematic since the fluorophore tumbles rapidly on its flexible chain.
The present invention overcomes many of the shortcomings associated with labeling nucleic acids. The present invention allows a pyrene, anthracene, phenanthrene, stilbene, tetracene or pentacene-derivatized nucleoside to be inserted within a DNA or RNA strand at any position and remain rigidly stacked within the helix. Because the fluorescent part of the label is situated as if it were a DNA base, the fluorescent groups are stacked neatly in the helix, and if placed adjacent to each other, interact with each other strongly, allowing for intense excimer emission.
The fluorescent nucleoside analogs of the present invention do not photobleach rapidly, making the labeled sample much longer lived and allowing the opportunity for measurements and study over a longer period. Moreover, since the preferred embodiment of the invention provides for .alpha.-linkages to the fluorescent moiety, quenching by adjacent .beta.-linked DNA bases is minimized. In addition, the direct attachment of the fluorescent moiety to the sugar residue in the nucleotide chain eliminates the flexible linker typically present in fluorescently labeled nucleic acids. This feature simplifies measurements such as time-resolved fluorescence anisotropy.